Trans-synaptic David

regulation of gene expression

D. Ginty, Hilmar

Bading and Michael

E. Greenberg

Harvard Medical School, Boston, Massachusetts, Neurotransmitters cascades nucleus

that

regulate transmit

the

of the postsynaptic

gene expression signal

from

cell. Ca2+

messengers

that regulate gene expression

The

and

Ca2+

CAMP

signals

induce

termed immediate early genes, within activation.

the expression have elucidated

of late response mechanisms

and CAMP signals

Current

in response expression

genes. The by which

Tram-synaptic regulation of gene expression is critical for neural development and for long-term adaptive changes in the mature nervous system. Neurotransmitters released from presynaptic cells regulate gene expression in postsynaptic cells by binding and activating specific postsynaptic receptors. The activation of neurotransmitter receptors leads to the stimulation of second messenger systems which transduce the signal to the nucleus where expression of specific genes is regulated. Of the many genes whose transcription is regulated by neurotransmitters, the C-$X proto-oncogene is the best characterized [ 11. It is a member of a family of genes, termed immediate early genes (IEGs), whose basal expression in unstimulated cells is low and whose transcription is rapidly and transiently induced by a wide range of extracellular stimuli in both neuronal and nonneuronal cells. Induction of IEGs occurs independently of new protein synthesis and is believed to be mediated via the post-translational modification of proteins already existing within the cell. Other IEGs whose transcription is activated by neurotransmitters include ZzJr.268, c-jun, junB and ~~0-77. In addition to inducing IEGs, neurotransmitters can also rapidly stimulate the expression of a variety of neuron-specific genes including those encoding vasoactive intestinal polypeptide (VIP), somatostatin, and proenkephalin [ 2-41. Many IEGs, including c-fos, encode transcription factors which are proposed to regulate the subsequent transcriptional activity of other genes, termed late response genes. Along with other members of the jii family, C-&S heterodimerizes with members of the jun family of DNAbinding proteins [ 51. Foy/jun heterodimers bind to API binding sites (consensus, TGAC/GTCA) in the regula-

to the

to neurotransmitters. a class

of genes, receptor

results

of

factors that regulate of recent experiments

neurotransmitter-induced

in Neurobiology

Introduction

messenger

membrane

of neurotransmitter

transcription

regulate immediate

Opinion

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plasma

and CAMP are two of the second

minutes

Many of these genes encode

through

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USA

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early gene expression.

1992, 2:312-316

tory regions of late response genes and promote their transcription. Neuron-specific late response genes that appear to be regulated by fo~‘jun complexes include those encoding tyrosine hydroxylase, nerve growth factor, and transin [Gs] To understand the importance of neurotransmitter-regulated gene expression in the nervous system the following must be determined: first, the mechanisms by which neurotransmitter-induced second messenger systems induce IEG expression (and also the genes that encode certain neurotransmitters and neuropeptides); second, the mechanisms by which IEGs, such as c-fos, regulate the expression of late response genes; and third, the role that IEGs and late response genes play in adaptive responses of the nervous system. Although all three of these topics are important for understanding how neurotransmitters elicit physiological changes in neurons, this review will focus on how neurotransmitters regulate IEG expression, and the role that the second messengers CAMP and Ca2 + play in this process. CAMP regulation

of transcription

Neurotransmitters such as dopamine and adenosine can regulate the activity of certain postsynaptic cells by modulating the cytoplasmic levels of CAMP. These neurotransmitters bind to specific receptors on postsynaptic membranes that activate membrane-bound GTP-binding proteins (G proteins). In a subset of neurons, the activated G proteins dissociate and then activate adenylate cyclase, which catalyzes the synthesis of CAMP. In most cases, the effects of increased cytoplasmic levels of CAMP are then mediated through activation of the cAMP-dependent protein kinase, protein kinase A (PKA). PKA is an enzyme that catalyzes the transfer of a phosphate group

Abbreviations element; C&K-Ca2+/calmodulin-dependent protein kinase; CaRE-Ca2+- response element; CRE-CAMP-response CREB-CAMP-response-element binding protein; CREM-CAMP-responsive element modulator; C protei+GTP-binding protein; IEG-immediate early gene; NMDA-N-methyl-o-aspartate; PKA-protein kinase A; SRE-serum-response element; VIP-vasoactive intestinal polypeptide.

312

@ Current

Biology

Ltd ISSN 0959-4388

Tram-synaptic

from ATP to serine or threonine residues on specific protein substrates within the postsynaptic cell [9,10]. When CAMP binds to the regulatory subunit of PKA, the holoenzyme dissociates to yield a regulatory subunit dimer and two active catalytic subunits. A variety of experiments have recently demonstrated that increased activity of the catalytic subunit is necessary for cAMP-induced activation of gene expression [3,11-131. In addition, other recent studies have shown that, once released from the regulatory subunit, the free catalytic subunit translocates from the cytoplasm to the nucleus where it phosphotylates nuclear factors involved in transcriptional control [ 14,151. Analysis of cAMP-responsive genes, including c-fos and the somatostatin gene, has revealed specific sequences in their promoter regions that are necessary and sufficient for cAMPinduced transcription. In the somatostatin gene, Montminy and colleagues [3] have identified a c&VIP-response element (CRE) located approximately 40 base pairs upstream of the transcriptional start site. A similar CRE is found in the promoter region of several other cAMP-responsive genes including c-fa, ZifJ268, proenkephalin and vasoactive intestinal polypeptide (VIP) genes [2,4,16,17], suggesting that this element is involved in the regulation of many CAMP-m sponsive genes. Subsequent to the discovety of the CRE, a protein that specifically binds to this sequence was purified and its gene cloned. This protein, the CAMP-m sponse element binding protein (CREB), appears to be constitutively bound to the CRE [ 18-201. However, when cells are exposed to agents that stimulate an increase in CAMP metabolism, CREB becomes newly phosphorylated on a specific residue, Ser133 [20]. This phosphorylation event activates CREBs ability to stimulate transcription. As CREB (Ser133) is an excellent substrate for purified PKA in vitro, it is believed that CREB is phosphorylated directly by PKA in viuo, rather than via an intermediate kinase. Taken together, these experiments suggest that elevated levels of CAMP trigger the activation of the catalytic subunit of PKA, which translocates to the nucleus and phosphotylates CREB. Once phosphorylated, CREB potentiates the transcription of genes containing a CRE within their regulatory regions. PKAmediated phosphorylation of CREB at Ser133 appears to be required for CREB to induce gene expression, as mutation of Ser133 to an alanine blocks CREBs ability to turn on transcription. CREB mutants containing a negatively charged amino acid at position 133, rather than a phosphorylated serine, do not activate CREmediated transcription in the presence or absence of cAMP [20]. A model has therefore been proposed whereby some aspect of the phosphate group on Ser133, in addition to its negative charges, is responsible for inducing a conformational change in CREB so that its transactivator domain becomes available to interact with other transcriptional regulatory proteins [201. It is also possible that events in addition to the phosphorylation of Ser133 are required to induce transcriptional activity of CREcontaining promoters. Several other proteins, in addition to CREB, have been identified that bind to CREs and regulate transcription. These proteins include other members of the CREB family, such as ATFl [21], and members of the AP

regulation

of gene expression

Cinty,

Bading and Greenberg

1 transcription complex such as junD and JunB [4]. While these proteins can bind to CRElike sequences and have been implicated in cAMP-dependent regulation of gene expression, their roles in neurotransmitterinduced transcription remain unclear. As many of these factors have the ability to homo- as well as heterodimerize, there is a potential for different combinations of factors to bind to the CRE and modulate transcription. Recent experiments have identified a factor, CAMP-m sponsive element modulator (CREM), which has the ability to bind the CRE as a CREB/CREM heterodimer. It has been proposed that, in cells expressing CREM, a CREMCREB heterodimer may negatively regulate CAMP[ 221. induced transcription In addition to the CRE, other structurally distinct CAMPresponse elements have been identified within regulatory regions of several cAMP-responsive genes [23*,24,25]. Recently, one such element within the c-fospromoter was shown to bind the transcription factor rNFIL-6 [23*]. This factor is a leucine zipper-containing protein with a high degree of homology to the transcription factor C/EBP. rNFIL-6 becomes phosphotylated and translocates to the nucleus in response to agents that increase intracellular levels of CAMP. In addition, expression of rNFIL-6 in NIH3T3 cells induces transactivation of c-fos gene constructs containing a C/EBP binding site [23-l. Taken together, these experiments suggest that an alternative pathway by which CAMP triggers gene induction may be through the phosphotylation and activation of rNFIL-6. Ca* + regulation

of transcription

Many neurotransmitters control the activity of postsynaptic cells by regulating cytoplasmic levels of Ca2+. Some neurotransmitters control the intracellular Ca2+ concentration by regulating membrane potential, thereby indirectly controlling the activity of voltage-sensitive Ca2+ channels. An example of a neurotransmitter that may function in this way is acetylcholine. The binding of acetylcholine to nicotinic cholinergic receptors leads to membrane depolarization and a Ca2+ influx through the voltage-sensitive Ca2+ channels. Recent experiments have established the importance of these channels in the control of gene expression in PC12 cells and cortical neurons [ 26281, Another class of neurotransmitter regulates intracellular Ca2+ metabolism through activation of phospholipase C activity and phosphatidylinositol metabolism. In the nervous system, this type of signalling pathway is activated by the interaction of glutamate with a subtype of the metabotropic glutamate receptor, termed mGLUR1 [29,30]. It remains to be determined whether activation of the metabotropic receptor is coupled to the activation of gene expression. In other systems, however, agents that activate phospholipase C activity are potent activators of IEG transcription, Another way in which neurotransmitters enhance the cytoplasmic levels of Ca2+ is by binding and activating receptors that are themselves ligand-gated Ca2+ channels. The best characterized receptor/Caz+ channel is the Nmethyl-D-aspartate (NMDA) receptor, a type of glutamate receptor that has been implicated in activity-dependent

313

314

Signailing mechanisms

neuronal plasticity [31*]. Several recent studies indicate that activation of the NMDA receptor leads to rapid induction of IEGs [ 32-351. While the pathways leading to IEG stimulation by NMDA-receptor activation are not clear, recent studies raise the possibility of the involvement of a protein tyrosine kinase [36,37]. To define the mechanisms by which enhanced cytoplas mic levels of Ca2+ trigger activation of IEG expression, initial studies employed PC12 cells, a rat pheochromocytoma cell line [38*,393. In these cells, nicotinic agonists and membrane depolarizing concentrations of KC1 induce c-& transcription [ 26,271. This effect is dependent on the influx of extracellular Ca2+ through dihydropyridine-sensitive Ca2+ channels as well as on the activity of the intracellular Ca2+ binding protein, calmodulin [ 26,271. Recent observations indicate that an element in the c-fm promoter, previously described as a CRE, also functions as a Ca2+ -response element (CaRE) [39]. This element, located 60 base pairs upstream of the initiation site of c-fos mRNA synthesis, binds CREB and has the ability to mediate transactivation of the c-fos gene in response to Ca2+ inllux as well as cAh4P [39]. In addition, phosphotylation of CREB at Ser133 is required for both Ca2+- and CAMPinduced transactivation of c-fm [38-l. As membrane depolarization has little or no effect on CAMP metabolism in PC12 cells [39,40], it is unlikely that PKA activation leads to CREB phosphotylation in response to Cal+ influx. A possible involvement of Ca2+/calmodulin-dependent protein kinases in this pathway is suggested by the finding that CREB is an excellent in vitro substrate for the multifunctional Ca2+/calmodulindependent protein kinase (CaMK) [38*,41]. Taken together, these expenmerits support a model whereby Ca2+ intlux regulates CREmediated c-fos transcription through activation of CaMK, which in turn phosphorylates and increases the activity of CREB. The presence of CREs in the upstream regulatory regions of a number of other depolarizationresponsive genes such as ZzJ268, and the proenkephalin and VIP genes [2,4,17] suggests that the Ca2+ signalling pathway that has been delined for c-fos may have a more general function. Several experiments indicate that, in addition to stimulating gene expression through the CREB pathway, Ca2+ may also control transcription by activating other transcription factors that bind to distinct regulatory elements. For example, c-fa gene constructs in which the CaRE/CRE is mutated, but that have the rest of the c-fa promoter intact, are still responsive to depolarizing stimuli [39]. These results indicate that c-fm elements, in addition to the CaRE/CRE, may also participate in Ca2+-induced c-fm transcription. One such element, the serum-response element (SRE), is centered 310 nucleotides upstream of the transcriptional start site of the c-fa gene. The SRE has previously been demonstrated to be required for growth-factor induced c-fm expression [42]. Observations from this laboratory indicate that the SRE may also mediate Ca2+ -induced c-fos expression (M Sheng and M Greenberg, unpublished data). Recent studies using macrophage and libroblast cell lines have identified another level at which Ca2+ may act to control c-fa expression [43*,44]. These exper-

iments have provided evidence for a Ca2+ -dependent block of transcriptional elongation. Using nuclear runon transcription analysis it has been shown that prior to stimulation, clfos transcription is initiated to some extent, but mRNA elongation is blocked within the first intron of the c-f@ gene. Relief of this elongation block requires Ca2+ and is achieved by many different stimulatory agents including dexamethasome, activators of PKA, activators of protein kinase C, and a Ca2+ ionophore. Together with enhanced transcriptional initiation, relief of the elongation block leads to induction of clfos mRNA levels. At present, it is unknown whether this Ca2+ -dependent elongation block plays a role in neurotransmitter regulation of c-fm expression in neurons. Ca*+

and CAMP crosstalk

It is becoming increasingly clear that Ca2+ and CAMP signals regulate gene expression in ways that are not completely independent. As mentioned above, Ca2+ and CAMPcan use at least one common intermediate, CREB, in regulating transcription. Although membrane depclarization and Ca2+ influx does not influence CAMP metabolism in PC 12 cells [ 39,401, membrane-depolarization induced IEG expression is attenuated in PC12 cells deficient in PKA [45]. Thus, pathways used by Ca2+ and CAMP to regulate IEG transcription are, at least in part, dependent on common intermediates. Interestingly, when cells are co-treated with agents that increase the cytoplasmic levels of both CAMPand Ca2 + , a greater than additive increase in transcription mediated by the c-fos CaRE/CRE is observed [39]. This finding is difficult to understand if both treatments work exclusively through phosphorylation of CREB at Ser133 and induction of CREB’s transactivator ability. Thus, it is reasonable to suggest that one or both of these signalling pathways may also regulate unique intermediary events in the control of transcription in neurons. Ca2+ and CAMPsignalling pathways have been found to interact at several additional levels in neurons. One such interaction is at the level of the Ca2+ channel. Agents that increase CAMP levels and PKA activity can inlluence the activity of voltage-sensitive Ca2+ channels [46]. For example, activation of dopamine Dl receptors results in a CAMP- and PKA-mediated increase in the activity of a dihydropyridine-sensitive Ca2+ current in bovine chroma& cells [47]. Therefore, neurotransmitters that activate gene expression via the cAMP-dependent pathway may also regulate gene expression by indirectly modulating Ca2+ influx. Under other circumstances, intracellular Ca2+ levels can modulate CAMP levels, which may then regulate gene expression via the PKAdependent pathway. In certain neurons, such as pyramidal and granule cells of the hippocampus, a Ca2+ -activated adenylate cyclase is expressed at high levels [48]. In these cells, glutamate-induced NMDA receptor activation results in a Ca2+ influx followed by an accumulation of CAMP [49]. Although not yet documented experimentally, it is reasonable to suggest that the elevated levels of CAMP might activate PKA-dependent pathways of gene expression.

Tram-synaptic

Crosstalk may also occur between signalling pathways at the level of the protein kinase. A model has been proposed whereby Ca2+ and CAMP signals converge on PKA [50]. In this model, Ca2+ is proposed to activate a protease, calpain, which may degrade the regulatory subunit of PKA and thereby lead to increased levels of its active catalytic subunit. This model predicts that Ca2+ and cAh4P converge to produce a prolonged sustained increase in PKA activity, greater than that produced by CAMPalone. As described above, the CAMPand Ca2+ signalling path ways also converge at the level of transcription factor phosphotylation. While CREB is known to be phosphotylated at the same site by both Ca2+ and CAMP pathways, it is reasonable to speculate that, under some circumstances, these two pathways might lead to phosphorylation at different sites on the same transcription factor. The phosphotylation of distinct sites on a single molecule might allow the two signalling pathways, when stimulated simultaneously, to have synergistic or antagonistic effects on transcription factor activity. Conclusions Although much progress has been made towards an understanding of the molecular mechanisms by which neurotransmitters control gene expression, many questions remain unanswered. The identification of CREB as a bifunctional target of Ca2+ and CAMP signalling pathways is only a first step in the characterization of a complex network of neurotransmitter-regulated signalling events. Clearly, other Ca2+ and cAMP-regulated transcription factors that bind to novel response elements remain to be identified. Additionally, the mechanisms by which second-messenger regulated transcription factors interact with the basal transcription complex remain to be elucidated. For example, how CREB phosphorylation influences the activity of RNA polymerase II is not yet understood. Although not addressed in this review, the elucidation of the function of IEGs is also critical for a full appreciation of the important role that neurotransmitter regulation of gene expression plays during neural development and in the physiological responses of mature neurons. In particular, it will be critical to identify late response genes whose expression is controlled by neurotransmitter-regulated IEGs. The characterization of these genes will greatly enhance our understanding of the mechanisms by which neurotransmitters regulate long-term adaptive changes of the nervous system. References

and recommended

Papers of particular interest, published view, have been highlighted as: . of special interest .. of outstanding interest 1.

SHENG M, GREENBERGME: The

2.

FINK JS, VERHAVEI M, WPER GOODMAN RH: The CGTCA

regulation of gene expression Cinty,

for Biological Activity of the Vasoactive Intestinal Peptide Gene cAMP-ReguIated Enhancer. f+oc Natl Acad Sci USA 1988, 856662-6666. 3.

MONT~UNYMR. SEVAR~NO K4 WAGNERJA, MANDELG, GODMAN RH: Identification of a Cyclic-AMP Responsive Element Within the Rat Somatostatin Gene. Proc Nat1 Acad Sci USA 1986, 83~2-6686.

4.

KOBIEBKI IA, CHU H-M, TAN Y, COMB MJ: cAMPDependent Regulation of Proenkephalin by JunD and JunB: Positive and Negative Effects of AP-1 Proteins. PYOCNat1 Acad Sci USA 1991, 88:10222-10226.

5.

~ONETIS TD, GEORGOPOULOS K, GREENBERG ME, LEDER P: c-jun Diierizes with Itself and with c-fos, Forming Complexes of Diierent DNA Binding AIBnities. Cell 19B8, 55:917-924.

6.

HENGERER B, LINDHOLMD, HEUMANNR, RUTHER U, WAGNER

EF, THOENEN H: Lesion-Induced Increase in Nerve Growth Factor mRNA is Mediated by c-fos. Proc Nat1 Acad Sci IJSA 1990, 87:389+3903. 7.

Regulation c-fos and Other Immediate Early Genes System. Neuron 1990, 4:477-485.

T, MANDEL G, Motif is Essential

Sequence

Differentiation

in

PC12

8.

GIZANG-GINSBERG E, ZIPP EB: Nerve Growth Factor Regulates Tyrosine Hydroxylase Gene Transcription Through a Nucleoprotein Complex that Contains c-fos. Genes Dezj 1990. 41477491.

9.

TAV~.ORSS: CAMP-Dependent 1989, 2648443-8446.

10.

MCKNIGHTGS: Cyclic AMP Second Qbin Cell Biol 1991, 3~213-217.

11.

GROW JR, PRICE DJ, GOODMAN HM, AVR~JCHJ: Recombinant Fragment of Protein Kinase Inhibitor Blocks Cyclic AMPDependent Gene Transcription. Science 1987, 238:53@533.

12.

MELLONPL, CLEGG CH, CORRELLLA, MCKNIGHTGS: Regulation of Transcription by Cyclic AMP-Dependent Protein Kinase. Proc Nat1 Acad Sci USA 1989, 86:48874891.

13.

DAY RN, WAIDER JA, MAURER RA: Protein finase Inhibitor Gene Reduces Both Basal and MultihormonalStimulated Prolactin Gene Transcription. J Rio1 Cbem 1989, 264431436.

14.

MEINKOTHJL, JI Y, TAYLORSS, FERAMISCOJR: Dynamics and Distribution of Cyclic AMP-Dependent Protein Kinase in Living Cells. Proc Nati Acad Sci USA 1990, 87~9595-9599.

15.

ADAMSSR, HAROOTUNIANAT, BUECHLERTJ, TAYLORSS, TSIEN RY: Fluorescent Ratio Imaging of Cyclic AMP in Single Cells. Nature 1991, 349694697.

16.

BERKOWITZ@ R~\GBOWOLKT, GIIMAN MZ: Multiple Sequence Elements of a Single Functional Class are Required for Cyclic AMP Responsiveness of the c-fos Promoter. Mel Cell Rio1 1989, 9:42724281.

17.

CHANCELIANPS, PENC P, KING TC, M~LBRANDT J: Structure of the NGFl-A Gene and Detection of Upstream Sequences Responsible for its Transcriptional Induction by Nerve Growth Factor. Proc Nat1 Auzd Sci USA 1989, 86:377-381.

18.

YA~IAMOTOKK, GONZALEZ GA, BIGCS WH, MONTMINY MR: Phosphorylation-Induced Binding and Transcriptional Efficacy of Nuclear Factor CREB. Nature 1988, 334:494-498.

19.

MONTMINYMR, BILEZIKJ~AN LM: Binding of a Nuclear Protein to the Cyclic AMP Response Element of the Somatostatin Gene. Nature 1987, 328:17%178.

20.

GONVUEZ

and Function of in the Nervous

S,'TSUKADA

MACHIDA CM, ROD~ANDKD, M~STRISIANL, MAGUN BE, CIMENT of the Gene Encoding the Protease G: NGF Induction

Transin Accompanies Neuronal CeIIs. Neuron 1989, 2:1587-1596.

reading

within the annual period of re-

Bading and Creenberg

Protein

Kinase.

Messenger

J Biol Cbem Systems.

Curr

GA, MONTMINYMR: Cyclic AMP Stimulates SOmatostatin Gene Transcription by Phosphorylation of CREB at Serine 133. Cell 1989, 59:675%%0.

315

316

Signalling

mechanisms

21.

HURSTHC, Tom

NF, JONES NC: ldentilication and Functional Characterisation of the Cellular Activating Transcription Factor 43 (ATF-43) Protein. Nucleic Acids Res 1991, 19:46014609.

22.

FOULKES NS, BORRELLI E, SASSONE-CORSIP: CREM Gene: Use of Alternative DNA-Binding Domains Generates Multiple Antagonists of CAMP-Induced Transcription. Cell 1991, 64739749.

the C/EBP-Related METZ R, ZIFF E: CAMP Stimulates Transcription Factor rNFIL-6 to Translocate to the Nucleus and Induce c-fos Transcription. Genes Dev 1991, 51754-1766. This study demonstrates that NFIL-6, a C/EBP-like factor, becomes phosphotylated and also translocates to the nucleus upon treatment of cells with agents that increase CAMP metabolism. Furthermore, NFIL6 is shown to enhance transcription of c,fi genes containing NFIL-6 binding sites in cotransfection assays. 23. .

24.

25.

26.

SHENGME, THOMPSON MA, GREENBERGME: CREB: a Ca2+ Regulated Transcription Factor Phosphorylated by CaM Kinases. Science 1991, 252:1427-1430. This study provides evidence that CREB is a bifunctional transcription factor that is a nuclear target for both CAMP- and Ca2+ -triggered second messenger pathways.

38.

.

SHENG M, MCFADDEN G, GREENBERG ME: Membrane Depolarization and Calcium Induce c-fos Transcription via Phosphorylation of Transcription Factor CREB. Neuron 1990, 4:571-582.

40.

HARTIGE, IQNCAREVIC IF, BUSCHERM, HEKRLICHP, RAHMS~RF HJ: A New CAMP-Response Element in the Transcribed Region of the Human c-fos Gene. Nucleic Acids Res 1991, 19:415%4159.

VAN NGI~YENT, KOBIERXI L, COMB M, HYMANSE: The Effect of Depolarization on Expression of the Human Proenkephalin Gene is Synergistic with CAMP and Dependent Upon a CAMP-Inducible Enhancer. J Neurosci 1990, 10:2825-2833.

41.

FISCH TM, PRYWESR, SIMON MC, ROEDEWRG: Multiple Sequence Elements in the c-fos Promoter Mediate Induction by CAMP. Genes Deu 1989, 3:19%211.

DA.SH PK, KAR. KA, Corrcos MA, PROVES R, KANDELER: CAMP-Response Element-Binding Protein is Activated by Caz+/Calmodulin- as well as CAMP-Dependent Protein Kinase. Proc Nat1 Acad Sci USA 1331, 88:5061-5065.

42.

KIVERAVM, GREE~ERG ME: Growth Factor-Induced Gene Expression: the Ups and Downs of c-fos Regulation. New Biol 1990, 2:751-758.

GREENBERGME, ZIFF EB, GREENE LA: Stimulation of Neuronal Acetylcholine Receptors Induces Rapid Gene Transcription. Science 1986, 234:8@83. MORGANJI, CURRANT: Role of Ion Fluxes in the Control c-fos Expression. Nature 1986, 322:552-555.

28.

MUR~HV TH, Worum PF, BAKABANJA: L-Type Voltage-Sensitive Calcium Channels Mediate Synaptic Activation of Immediate Early Genes. Neuron 1991, 7:625-635.

29.

Mksu M, TANABEY, TXJCHIDA K, SHIGEMOTO R, NAKANISHI S: Sequence and Expression of a Metabotropic Glutamate Receptor. Nutttre 1991, 349:76G765.

of

HOIJAMEI) KM, KLIIJPERJL, GLBEKTTL, HALDEMANBA, O’HARA ER, ALMER?W, HAGEN FS: Cloning, Expression PJ, MULVIHILL and Gene Structure of a G Protein-Coupled Glutamate Receptor from Rat Brain. Science 1991, 252:131%1321.

MADISONDV, SCHUI~ EM: LTP, Post or Pre? A Look at the Evidence for the Locus of Long-Term Potentiation. Neuj Biol 1991, 3:54?557. This article briefly reviews electrophysiological a5pect.s of long-term pot tentiation (LTP) and summarizes recent studies on cellular mechanisms that may be important for the induction, maintenance and expression of LTP.

43. .

COL~ART MA, TO~JRKINE N, BELIN D, VASSAIU P, JEXNTEUR P, BWVCHARDJ-M: c-fos Gene Transcription in Murine Macrophages is Modulated by a Calcium-Dependent Block to Elongation in lntron 1. Mol Cell Biol 1991, 11:28262831. This study provides evidence for the presence of a block to transcriptional elongation within the first intron of the murine c-&s gene. Relief of this block occurs when cfas is induced by a variety of extracellular stimuli 44.

MECHTI N, PIECHACZM(M, BIANCHARDJ-M, JEANTEIJKP, LEBLEU B: Sequence Requirements for Premature Transcription Arrest Within the First lntron of the Mouse c-fos Gene. Mel Cell Biol 1991, 11:2832%2841.

45.

GINS DD, GLOWACKAD, BADERD, HIDAKAH, WAGNERJ: Membrane Depolarization and Ca2+ Influx Require CAMP-Dependent Protein Kinase for Induction of Immediate Early Genes in PC12 Cells. J Biol Cbem 1991, 26617454-17458.

46.

CATTE~ALL WA: Structure and Function Ion Channels. Science 1988, 243:5&61.

47.

AJITALEJO CA, ARlANoMA, PERIL

48.

Xu 2, REFSIM CD, MERCHANTKM, DORA DM, STORMDR: Distribution of mRNA for the Calmodulin-Sensitive Adenylate Cyclase in Rat Brain: Expression in Areas Associated with Learning and Memory. Neuron 1991, 6:431-443.

49.

CHETKOXH DM, GRAY R, JOHNSON D, Sm’rr JD: N-MethylD-Aspartate Receptor Activation Increases CAMP Levels and Voltage-Gated CaZ+ Channel Activity in Area CA1 of Hippocampus. Proc Nat1 Acad Sci USA 1991, 88:64674471.

50.

ASZODI A, MULLERU, FR~EDRICHP, SPATZ HC: Signal Convergence on Protein Kinase A as a Molecular Model of Learning. Proc Natl Acad Sci USA 1991, 88~5832-5836.

31. .

32.

33.

34.

O’DELLTJ, KANDELER, GRANT SG: Long-Term Potentiation in the Hippocampus is Blocked by Tyrosine Kinase Inhibitor. Nature 1991, 353:55%560.

39.

27.

30.

37.

SZEKELYAM, BARBACC~AML, ALHO H, COSTA E: In Priiary Cultures of CerebeIlar Granule Cells the Activation of NMethyl-D-Aspartate-Sensitive Glutamate Receptors Induces c-fos mRNA Expression. Mel Pbarmacol 1989, 35:401408. SZEKELYAM, COSTA E, GRAYSONDR: Transcriptional Program Coordination by N-Methyl-D-Aspartate Sensitive Glutamate Receptor Stimulation in Primary Cultures of Cerebellar Neurons. Mol Pbarmacol 1990, 38:624-633. COLE AJ, SMFEN DW, BAKABAN JM, WOKLEYPF: Rapid Increase in an Immediate Early Gene Messenger RNA in Hippocampal Neurons by Synaptic NMDA Receptor Activation. Nature 1989, 34034741i76.

35.

WISDEN W, ERRINGTONML, WIIJ~~MS S, D~INN~T SB, WATERS C, HITCHCOCK D, EVAN G, Buss TVP, HUNT SP: Differential Expression of Immediate Early Genes in the Hippocampus and Spinal Cord. Neuron 1990, 4:603414.

36.

BALXNG I-l, GREENBERG ME: Stimulation of Protein Tyrosine Phosphorylation by NMDA Receptor Activation. Science 1991, 253:912-914.

of Voltage-Sensitive

RL, Fox AP: Activation of Facilitation Calcium Channels in Chromaflin Cells by Dl Dopamine Receptors Through a CAMP-Dependent Mechanism. Nature 1990, 348:23%242.

DD Ginty, H Bading and ME Greenberg, Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.